The goal of this project is to identify chemical probes that inhibit phosphopantetheinyl transferase (PPTase) of bacterial secondary metabolism. This enzyme governs a posttranslational modification event that is essential to activation of polyketide and non-ribosomal peptide biosynthetic machinery. Many examples within these natural product classes have been identified as bacterial virulence factors required for pathogenesis. A competitive inhibitor or inactivator of PPTase activity could serve as potential therapeutic lead against a variety of Gram negative and Gram positive pathogens, including, among others, Mycobacterium tuberculosis, Staphylococcus aureus, and Escherichia coli. The essential nature of PPTase to fatty acid biosynthesis has targeted it for antimicrobial therapeutic development, and several inhibitor classes have been identified in the literature. However, these molecules do not show activity against PPTases of secondary metabolic pathways, a major target for drug discovery. We have developed a FRET-quench HTS bioassay for PPTase activity that functions in microplate format. This fluorescence-based system meets the minimal liquid-handling requirements of the NCGC, and it has been demonstrated against a small panel of PPTase inhibitor analogs. When identified, hits elucidated from this screening effort will serve as leads for antimicrobial therapeutic discovery. Furthermore, these compounds will also serve to further our ongoing NIH-funded investigations into the biosynthetic mechanisms of modular synthases. [unreadable] [unreadable] [unreadable]